Preclinical evaluation of new GRPR-antagonists with improved metabolic stability for radiotheranostic use in oncology

Background The gastrin-releasing peptide receptor (GRPR) has been extensively studied as a biomolecular target for peptide-based radiotheranostics. However, the lack of metabolic stability and the rapid clearance of peptide radioligands, including radiolabeled GRPR-antagonists, often impede clinical application. Aiming at circumventing these drawbacks, we have designed three new GRPR-antagonist radioligands using [99mTc]Tc-DB15 ([99mTc]Tc-N4-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt; AMA: p-aminomethylaniline; DIG: diglycolate) as a motif, due to its high GRPR-affinity and stability to neprilysin (NEP). The new analogues carry the DOTAGA-chelator (1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid) through different linkers at the N-terminus to allow for labeling with the theranostic radionuclide pair In-111/Lu-177. After labeling with In-111 the following radioligands were evaluated: (i) [111In]In-AU-SAR-M1 ([111In]In-DOTAGA-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt), (ii) [111In]In-AU-SAR-M2 ([111In]In-[DOTAGA-Arg]AU-SAR-M1) and (iii) [111In]In-AU-SAR-M3 ([111In]In-[DOTAGA-DArg]AU-SAR-M1). Results These radioligands were compared in a series of in vitro assays using prostate adenocarcinoma PC-3 cells and in murine models. They all displayed high and GRPR-specific uptake in PC-3 cells. Analysis of mice blood collected 5 min post-injection (pi) revealed similar or even higher metabolic stability of the new radioligands compared with [99mTc]Tc-DB15. The stability could be further increased when the mice were treated with Entresto® to in situ induce NEP-inhibition. In PC-3 xenograft-bearing mice, [111In]In-AU-SAR-M1 displayed the most favourable biodistribution profile, combining a good tumor retention with the highest tumor-to-organ ratios, with the kidneys as the dose-limiting organ. Conclusions These findings strongly point at AU-SAR-M1 as a promising radiotherapeutic candidate when labeled with Lu-177, or other medically appealing therapeutic radiometals, especially when combined with in situ NEP-inhibition. To this goal further investigations are currently pursued. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00242-6.


Background
Prostate cancer (PC) is the fourth most commonly diagnosed cancer worldwide and the second most common in men (Sung et al. 2021).PC incidence rates are notably higher in high development index countries, and are rapidly escalating in regions with increasing life expectancy too (Sung et al. 2021).There are currently three main tools for diagnosis of prostate cancer, prostate specific antigen (PSA) blood measurement, digital rectal examination and Gleason scoring of biopsy material.Gleason scoring is also useful for cancer staging and therapy planning (Faviana et al. 2021).Patient management and treatment options heavily rely on accurate detection, staging and spread assessment of the disease.Thus, diagnostic tools are needed which can provide results accurately, fast and non-invasively, thereby minimizing biopsy-associated human error and patient discomfort.An emerging field for diagnostic and therapeutic treatment of cancer is radiotheranostics, employing target-specific radionuclide carriers for diagnostic imaging with positron emission tomography (PET) (e.g.  or single photon emission computed tomography (SPECT) (e.g.often combined with computed tomography (CT), and for delivering radiotoxic doses to eradicate tumor lesions (e.g.(Dumont et al. 2013).
The gastrin-releasing peptide receptor (GRPR) represents a promising target in prostate cancer radiotheranostics.It is a member of the bombesin family of receptors and it is overexpressed in 63-100% of all prostate cancer cases while having a low expression level in healthy tissues of the body, with the exception of the pancreas and the gastrointestinal tract (Beer et al. 2012;Mansi et al. 2021;Patel et al. 2006;Rinne et al. 2021).It has been found that GRPR expression more clearly correlates to Gleason scoring than prostate specific membrane antigen (PSMA), with higher expression evident in earlystage prostate cancer and, most importantly, minimal expression in prostate hyperplasia (Faviana et al. 2021).Thus, diagnostic imaging of GRPR expression might be a valuable tool in the early discovery of prostate cancer, confined or already infiltrating in the surrounding tissues, providing more therapeutic options for patients.
Bombesin (BBN, Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 ), first isolated from the European fire-bellied toad Bombina bombina in 1971, is known for its high affinity for the GRPR (Anastasi et al. 1971).Bombesin analogues, particularly C-terminal peptide fragments, have extensively been investigated as templates for the development of radiotheranostics (Ferreira et al. 2017;Mansi et al. 2021;Moreno et al. 2016).Early efforts were focused on receptor agonists, because rapid internalization into tumor cells after receptor binding of such radioligands was believed to be crucial for high and sustained tumor uptake (Mansi et al. 2021).In practice, GRPR activation by agonists triggered acute adverse effects mainly in the gastrointestinal system and was also associated with mitogenic actions (Bodei et al. 2007;Rozengurt et al. 1990).Therefore, a shift in GRPR-radioligand development towards antagonists occurred following the trends in neuroendocrine tumor radiotheranostics based on somatostatin (Haider et al. 2020).Several studies have demonstrated that radiolabeled GRPR antagonists achieve higher tumor uptake and retention in comparison with agonists, despite their significantly inferior internalization in tumor cells (Cescato et al. 2008;Maina et al. 2017;Mansi et al. 2009;Mitran et al. 2020;Nock et al. 2005).Furthermore, their short amino acid chains, in combination with the structural changes introduced in the native sequence lead to enhanced resistance to degrading peptidases in the blood (Schottelius and Wester 2009).
Radiochemical purity was determined by reversed phase high performance liquid chromatography (RP-HPLC).The system was equipped with a LaPrep Sigma HPLC LP1100 pump (Hitachi High-Tech Corporation, Hitachinaka, Ibaraki, Japan) linked to a 40D LWL UV-detector with a 4 µL flow cell (Knauer, Berlin, Germany) and a Flow scan radioactivity detector (Bioscan, Paris, France) with an FC-3300 NaI/PMT radioactivity probe (Eckert & Ziegler, Berlin, Germany) and a manual sample injector 7725i (Rheodyne, Chrome Tech, Apple Valley, MN, USA,) fitted with a 20 µL sample loop (IDEX Health & Science, LLC, Rohnert Park, CA, USA).For data analysis and instrument monitoring the Open Lab EZChrome Elite software (Agilent, Santa Clara, CA, USA) was used.The column used was a Luna C18 column (5 µm, 100 Å, 150 × 4.6 mm from Phenomenex, Vaerløse, Denmark).For elution a two solvent gradient system was adopted, starting at 95% A/5% B up to 30% A/70% B at 15 min, going to 5% A/95% B at 17 min, remaining in this ratio for 2 min and returning to initial conditions over another 2 min (A: 0.1% trifluroacetic acid (TFA) in water; B: 0.1% TFA in acetonitrile, MeCN).
Radiometal-chelate stability was assessed by incubation of test radioligand (5 µL of the reaction mixture, 115 pmol) with a × 1000 molar excess of EDTA (115 nmol, 8.7 µL) for 1 h at room temperature.This result was compared to the same amount of radioligand left for 1 h at room temperature in an equivalent volume of PBS (8.7 µL).Samples were analysed by iTLC.

GRPR-specificity
For the determination of GRPR-specificity, PC-3 cells were seeded in 35 mm tissue-culture dishes (Corning ® , Corning, NY, USA) and test radioligand was added to each well (1 nM).In a subset of dishes, 1 µM of the unlabeled NOTA-PEG 2 -RM26 was added for blocking the GRPR.Cells were incubated for 1 h at 37 °C and 5% CO 2 , washed with PBS, dissociated, collected, and sample-radioactivity was measured using a 2480 WIZARD 2 automatic gamma counter (PerkinElmer ® , Waltham, MA, USA).Experiments were performed in triplicate along with three standards for each compound.

GRPR binding affinity and kinetics
For evaluation of the receptor affinity and binding kinetics, PC-3 cells were seeded in 100 mm Nunc ™ Petri dishes (Thermo Fisher, Waltham, MA, USA).A LigandTracer Yellow device (Ridgeview Instruments AB, Uppsala, Sweden) was used to measure the radiation from the Petri dish.Background with RPMI-1640 medium was measured for 30 min, after which 1 nM of radioligand was added and measured for 2 h.An additional concentration (3 nM) was measured for another 2 h, thereafter the medium was aspirated, replaced with fresh medium, and left running overnight to measure the dissociation rate.Data was fitted with the TracerDrawer software (Ridgeview Instruments AB, Uppsala, Sweden).

Cellular processing assay
PC-3 cells were seeded in 35 mm tissue-culture dishes.The next day, after aspiration of the medium and washing of the cells with PBS, 1 nM of test radioligand in complete growth medium was added to each dish and incubated at 37 °C in a 5% CO 2 atmosphere for predetermined time points.At each timepoint the medium was aspirated, the dishes were washed with PBS and incubated with acid wash buffer (500 µL, 0.2 M glycine buffer, 0.15 M NaCl, 4 M Urea, pH 2) for 5 min on ice (membrane-bound fraction).Then the cells were washed with PBS and a basic solution (500 µL, 1 M NaOH) was added; the dishes were left to incubate at 37 °C in 5% CO 2 for 30 min and scraped.Cell-lysates were collected (internalized fraction) and the activity of each fraction was measured on the gamma counter.The experiment was performed in triplicates.

Metabolic stability
The metabolic stability of radioligands was tested by HPLC analysis of mice blood collected 5 min post-injection (pi).For the experiment, healthy Swiss Albino mice (30 ± 5 g body weight; > 8 weeks of age) were purchased from the NCSR "Demokritos" Animal House (Institute of Biosciences & Applications, NCSR "Demokritos", Athens, Greece).Experiments were performed in accordance with the European guidelines in supervised and licensed facilities (EL 25 BIOexp 021) adhering to approved study protocols (Department of Agriculture and Veterinary Service of the Prefecture of Athens, protocol number #440,448, 01-06-2021).
Animals in control groups (n = 3) were injected intravenously (iv) with a bolus of test radioligand (100 μL, 2 nmol of peptide in PBS/EtOH v/v 9:1).Additional groups were pre-treated with a suspension of Entresto ® (200 µL, 12 mg of Entresto ® ) by oral gavage 30 min prior to iv radioligand injection.The suspension was prepared from 200 mg pills (containing 24 mg sacubitril/26 mg valsartan, Novartis AG, Basel, Switzerland) turned to fine powder in a mortar, suspended in tap water and distributed in equal animal-individualized doses in Eppendorf tubes.Five min pi, the animals were euthanized and blood was collected directly from the heart in a prechilled heparinized syringe.Blood samples were immediately mixed with EDTA (0.1 mM, 20 μL) in LoBind Eppendorf tubes on ice.The mixture was centrifuged at 2000 g for 10 min at 4 °C and the plasma was collected and diluted with an equal volume of MeCN.
After a second centrifugation at 15,000 g and 4 °C for 10 min, the supernatant was collected in a glass vial and concentrated under mild heating (50 °C) and a gentle stream on N 2 (to 50-100 μL).Physiological saline was then added to the samples (final volume of 450-500 μL) and the resulting solutions were filtered through Millex GV filters (0.22 μm, 13 mm diameter, Millipore, Milford, CT, USA).To determine the percentage of intact peptide, aliquots were analysed by reversed phase radio-HPLC.For analyses a Symmetry Shield RP-18 (5 μm, 3.9 mm × 20 mm) cartridge column (Waters, Eschborn, Germany) was eluted with the following gradient: starting at 100% A-0% B and reaching to 60% A-40% B in 40 min (A: 0.1% v/v aqueous TFA, B: MeCN).A Waters Chromatograph connected to a Gabi gamma detector (Raytest RSM Analytische Instrumente GmbH, Straubenhardt, Germany) was used in analyses controlled by the Empower Software (Waters, Milford, MA, USA).The peak corresponding to the intact peptide was identified by co-injection of an aliquot of the labeling solution with the corresponding blood samples.Experiments were performed in triplicate and presented as average % intact radioligand ± standard deviation (sd).

Biodistribution and SPECT/CT imaging
Animal experiments were planned and performed in accordance with national legislation on protection of laboratory animals, and protocols were approved by the Ethics Committee for Animal Research in Uppsala, Sweden (5.8.18-00473/2021).BALB/c nu/ nu female mice were xenografted by subcutaneous injection of PC-3 cells on the hind leg (7 × 10 6 cells/mouse).At the date of the experiment, approximately 6 weeks after implantation, tumour sizes reached 0.11 ± 0.05 g.Groups of four mice were used for each time point.In the first group, mice were iv injected with test radiopeptide (30 kBq in 100 µL PBS supplemented with 1% bovine serum albumin (BSA), corresponding to 40 pmol) and euthanized at 4 h pi.Tumors and relevant organs were collected, weighted and sample radioactivity was measured using the gamma counter.In the case of [ 111 In] In-AU-SAR-M1 two additional mice groups underwent the same procedure but were euthanized either at 24 h pi, or at 4 h pi following radioligand co-injection with excess (5 nmol) NOTA-PEG 2 -RM26 to determine the in vivo GRPR-specificity.For statistical analysis Two-Way Anova with Tuckey's post hock analysis was employed, using Graph-Pad Prism 7 for Windows ® (GraphPad Software, Boston, Massachusetts USA).
For SPECT/CT imaging, one mouse was injected with [ 111 In]In-AU-SAR-M1 (1.2 MBq in 100 µL of 1% BSA in PBS, 80 pmol); for imaging the labeling was conducted as previously described but at a 10 MBq/nmol molar activity.Image acquisition was performed at 4 h pi under anaesthesia and at 24 h pi after euthanasia with CO 2 asphyxiation.Whole body SPECT/CT scans were acquired using a NanoScan SPECT/CT instrument (Mediso Medical Imaging Systems, Budapest, Hungary).The acquisition time was 20 min.SPECT raw data were reconstructed using Tera-TomoTM 3D SPECT reconstruction technology (version 3.00.020.000;Mediso Medical Imaging Systems Ltd.).CT data were reconstructed using Filter Back Projection and fused with SPECT files with the Nucline 2.03 Software (Mediso Medical Imaging Systems Ltd.).The scans are presented as maximum intensity projections in the RGB colour scale.
When assessing their radiometal-chelate stability, the radiolabeled compounds remained stable in the presence of a 1000-fold excess EDTA and PBS within 1 h at room temperature.None of the radioligands showed a significant In-111 release after 1 h incubation in PBS when compared by iTLC directly post labeling.By comparing the stability in PBS and EDTA, results for [ 111 In]In-AU-SAR-M1 revealed no significant difference, while [ 111 In]In-AU-SAR-M2 and [ 111 In]In-AU-SAR-M3 showed a small, but significant difference in radiometal-chelate stability, which was more pronounced for [ 111 In]In-AU-SAR-M3 (Table 1).In vitro assays

GRPR-specificity
The radioligands displayed high and GRPR-specific uptake in PC-3 cells during 1 h incubation at 37 °C.In the presence of an excess of NOTA-PEG2-RM26, to block the GRPR sites on the cells, these values dropped to < 1.5% for all three radioligands (p < 0.0001, Fig. 3), in agreement with a GRPR-mediated process.

GRPR-binding affinity and kinetics
The equilibrium dissociation constant K D was determined by fitting the data to a 1:2 binding site model, which provided a superior fit over a 1:1 site model for

Metabolic study
As revealed by HPLC analysis of peripheral blood collected from healthy mice at 5 min pi, the three new radioligands showed similar metabolic stability with the [ 99m Tc] Tc-DB15 reference (Table 3).However, unlike [ 99m Tc]Tc-DB15, treatment of animals with Entresto ® significantly increased their stability, indicating the involvement of NEP in their degradation (Table 3).showed comparable stability.For the treated groups, [ 111 In]In-AU-SAR-M1 showed an equivalent stability to [ 99m Tc]DB15, while the other two radioligands showed a higher stability compared to the reference compound (p < 0.01 and p < 0.05, respectively).Unlike [ 99m Tc]DB15 which was shown to be NEP-resistant, the new analogues showed a significant increase in stability between the control and the Entresto ® treated groups, implying the involvement of NEP in their in vivo degradation.

Biodistribution and SPECT/CT imaging
The In-AU-SAR-M3 are summarized in Fig. 6a and Additional file 1: Table S1, while the tumor-to-organ ratios (T/O) are given in Fig. 6b and Additional file 1: Table S2.was chosen for further studies to determine in vivo GRPR-specificity of uptake at 4 h pi as well as to assess its biodistribution at 24 h pi (Fig. 7, Additional file 1: Table S1).
As shown in Fig. 7a, a significant decrease in pancreatic uptake (2 ± 1%IA/g controls vs. 0.18 ± 0.05%IA/g in block, p < 0.0001) as well as in tumor uptake (11 ± 1%IA/g controls vs. 0.8 ± 0.4%IA/g in block, p < 0.0001) was observed between the animals coinjected with excess of the GRPR-antagonist NOTA-PEG 2 -RM26 compared to controls, further implying a GRPR-mediated process.Kidney uptake was significantly higher in the blocked animals (3.1 ± 0.3%IA/g controls vs. 4.5 ± 0.6%IA/g in block, p < 0.01).By comparing the 4 h and 24 h biodistribution of [ 111 In]In-AU-SAR-M1, we firstly observe a rapid clearance from the GRPR-rich mouse pancreas with only 5% of initial uptake remaining at 24 h pi in this tissue.On the other hand, the tumor uptake decreased approximately by 40% within the same time frame (11 ± 1%IA/g at 4 h pi to 6.3 ± 0.7%IA/g at 24 h pi, p < 0.0001).The kidney uptake remained on the same level with a tendency to decrease over time (p > 0.05).
An increasing T/O ratio over time was established for almost all organs (Fig. 7b) with the exception of the kidneys.Tumor-to-blood showed a > threefold increase over time (from 160 at 4 h pi to > 550 at 24 h pi).The tumor-to-liver ratio remained approximately the same, while the tumor-to-muscle ratio increased by about 1.8-fold.The tumor-tobone ratio remained high (> 150) and increased only marginally at 24 h pi.The tumorto-pancreas ratio showed an excellent increase, approximately sixfold at 24 h pi (9 ± 11 to 57 ± 18), as consistent with a radioantagonist profile (Abouzayed et al. 2023;Damiana et al. 2023).
SPECT/CT imaging of [ 111 In]In-AU-SAR-M1 was conducted at 4 h and 24 h pi and representative images are included in Fig. 8. Tumors were well visualized in both timepoints along with the kidneys against a very clear background, in agreement with biodistribution results.

Discussion
Targeting of GRPR by means of peptide radionuclide-carriers has been regarded as a promising approach in the treatment of prostate cancer (Abouzayed et al. 2020;Dumont et al. 2013;Kurth et al. 2020;Mitran et al. 2019).Based on the GRPR overexpression in the early stages of the disease as opposed to healthy or hyperplastic surrounding tissue, GRPR-directed radiopharmaceuticals are expected to play a significant role in the management of prostate cancer, especially in primary and oligometastatic stage (Beer  Mansi et al. 2021;Patel et al. 2006;Rinne et al. 2021).The first-in-human study employing the therapeutic [ 177 Lu]Lu-RM2 radioligand to assess this approach, confirmed the excellent tolerability of the GRPR-antagonist.Moreover, over-expression of GRPR was reported in other cancers, among the most relevant for use of a GRPR targeting radiotherapy are gastrinomas (about 100% express GRPR), gastrointestinal stromal tumors (GIST, about 85%) and estrogen receptor positive breast cancer (over 80%) (Cornelio et al. 2007;Dimitrakopoulou-Strauss et al. 2007;Morgat et al. 2017;Reubi et al. 2004;Reubi et al. 2002).Clinical data demonstrates high uptake of GRPR-targeting antagonistic peptides in patients with estrogen receptor positive breast cancer both in primary lesions, lymph node and bone metastases (Chernov et al. 2023;Nock et al. 2021;Zang et al. 2018).
Yet, two major problems of GRPR-antagonist radioligands need to be resolved to maximise efficacy, to reduce pancreatic uptake and to enhance metabolic stability in circulation (Kurth et al. 2020).
Amongst the radiolabeled GRPR-antagonists developed in recent years (Mansi et al. 2021;Nock et al. 2023), [ 99m Tc]Tc-DB15 has especially attracted our attention by combining a high GRPR-affinity, resistance to NEP in the bloodstream, good tumor uptake and excellent tolerability in patients (Nock et al. 2021).In an attempt to reproduce these promising qualities in theranostics beyond SPECT/CT diagnostic imaging alone, trivalent radiometals were inevitably considered, and especially the In-111/Lu-177 theranostic pair.Toward this goal, the N-terminal N 4 of [ 99m Tc]Tc-DB15 was replaced by the DOTAGA-chelator in the first antagonist AU-SAR-M1 presented herein, inadvertently leading to the loss of a positive charge attributed to the [ 99m Tc]Tc(O) 2 (N 4 )] +1 radiometal-chelate (Nock et al. 2021).To counterbalance this loss, potentially impairing GRPR-affinity (Zhang et al. 2007), a basic L Arg or D Arg residue was introduced next to DOTAGA-chelator in AU-SAR-M2 and AU-SAR-M3, respectively.The impact of the above structural modifications on the respective [ 111 In]In-radioligands was assessed in a series of in vitro and animal assays, based on GRPR-expressing human cancer cells, to reveal the most promising candidate for further evaluation after labeling with Lu-177.Eventually, a well-characterized radiotheranostic pair for translation in prostate cancer patients could become available directly or via the new information acquired in this work.
After successful labeling with In-111, the three radioligands were tested in PC-3 cells to evaluate their cell uptake pattern and their GRPR-specificity.As it shown in Fig. 3, the radiotracers displayed high cellular association, which was clearly GRPR-driven.Furthermore, the conjugates retained a typical radioantagonist profile with the bulk of the cell-associated activity remaining bound on the cell membrane and slowly internalizing over time (Fig. 4).
The GRPR-affinity studies revealed a few interesting results (Table 2).First, [ 111 In]In-AU-SAR-M2 exhibited an order of magnitude higher receptor affinity than the other two analogues, a finding attributed to the positive charge of L-Arg next to DOTAGA (Zhang et al. 2007).Second, the real time measured affinities for [ 111 In]In-AU-SAR-M1 and [ 111 In]In-AU-SAR-M3 were found similar to the affinity of [ 111 In]In-DOTAGA-PEG 2 -RM26 (K D = 0.44 ± 0.05 nM) and its theranostic counterpart [ 177 Lu]Lu-DOT-AGA-PEG 2 -RM26 (K D = 0.4 ± 0.2 nM) (Mitran et al. 2019; Mitran et al. 2016).Third,  [ 111 In]In-AU-SAR-M2 and [ 111 In]In-AU-SAR-M3 displayed different dissociation constants, despite the presence of the positive charge next to DOTAGA in both due to the basic L/D Arg.This unexpected finding implies that the different 3D-configuration at the N-terminal of the peptide ( L Arg vs. D Arg) interferes with the interaction of the whole molecule with the GRPR.In the case of D Arg, the impact of such disadvantageous molecule configuration turned out to overwhelm the influence of the positive charge introduced by the basic residue (Table 2).Furthermore, the fact that [ 111 In]In-AU-SAR-M2 demonstrated only 1:1 interaction with GRPR, unlike [ 111 In]In-AU-SAR-M3 displaying 1:2 interaction (with very close rates of association and dissociation), corroborates this hypothesis.In fact, affinity data for [ 111 In]In-AU-SAR-M2, were analysed by a 1:2 model as well, but the K D1 found in the range of 10 -20 M turned out to be unrealistic for accurate measurements, leaving the 1:1 model as the only rational option.
According to the metabolic stability results in mice peripheral blood, [ 111 In]In-AU-SAR-M2 was as stable as [ 99m Tc]Tc-DB15, with [ 111 In]In-AU-SAR-M1 and [ 111 In]In-AU-SAR-M3 showing somewhat lower stability (Table 3).Unlike [ 99m Tc]Tc-DB15 previously shown to be NEP-resistant (Nock et al. 2021), the new radioligands showed significant stability increase in the blood of mice treated with Entresto ® in vivo releasing the potent and selective NEP inhibitor sacubitrilat.This finding implicates NEP in their in vivo degradation (Kanellopoulos et al. 2020).It appears that exchange of the radiometal-chelate from [[ 99m   [ 111 In]In-AU-SAR-M3 resulted in lower stability.The observed differences may represent distinct conformations of each molecule, leading to discrete ways of accessing the active centre of the enzyme, and their elucidation requires further dedicated studies.The different resistance of the new radioligands to NEP, as well as the degree of their stabilization by Entresto ® should be taken into account in view of treatment options.Radioligand stabilization applying a fully characterized registered drug, like Entresto ® , is expected to enhance their tumor-targeting capabilities, a highly desirable feature for targeted radiotherapy with a therapeutic radionuclide such as Lu-177 (Kanellopoulos et al. 2020;Nock and Maina 2019).
As expected, the higher GRPR-affinity and better metabolic stability of [ 111 In]In-AU-SAR-M2 translated in higher tumor uptake in PC-3 xenograft-bearing mice.On the other hand, [ 111 In]In-AU-SAR-M3 demonstrated the lowest tumor uptake (Additional file 1: Table S1), although its GRPR-affinity and in vivo stability were comparable with [ 111 In]In-AU-SAR-M1 (Fig. 3, Table 2, 3).Furthermore, [ 111 In]In-AU-SAR-M3 displayed the highest kidney uptake, but the lowest blood values and whole body retention of activity (Additional file 1: Table S1).These findings, taken together, indicate that receptor-affinity and metabolic stability, while crucial, are not the exclusive factors dominating the biodistribution profile.Among the three radioligands, [ 111 In]In-AU-SAR-M2 showed the highest uptake in small intestines and pancreas, both GRPR-rich organs (Körner et al. 2014;Reubi 2003), most probably as a result of its higher GRPR-affinity in comparison with the other two compounds.The tumor and pancreatic uptake were shown to be GRPR-specific during an additional GRPR-blockade study in the case of [ 111 In]In-AU-SAR-M1 (Fig. 7a).Overall, more favourable T/O ratios were achieved by [ 111 In]In-AU-SAR-M1 amongst the three radioligands and hence its biodistribution was additionally assessed at 24 h pi.The background activity declined from 4 to 24 h pi, including the GRPR-rich mouse pancreas and small intestines, but the kidney uptake remained at the same level.Tumor uptake dropped to 57% of the 4 h pi value.However, as the decrease of activity levels in other organs/tissues was faster (with the exception of the kidneys), T/O ratios increased, as typically observed for receptor antagonists (Lymperis et al. 2019).
Based on the above, [ 111 In]In-AU-SAR-M1 turned out to be a new promising diagnostic tool for diagnosis using SPECT/CT, combining a high tumor uptake with a very clear background at 4 h pi already.Furthermore, due to its good tumor retention and faster background clearance, [ 111 In]In-AU-SAR-M1 was able to provide excellent images at both 4 and 24 h pi, indicating the promising translation prospects to the respective therapeutic [ 177 Lu]Lu-AU-SAR-M1 counterpart.
The tumor-to-kidney ratios for [ 111 In]In-AU-SAR-M1 were 3.6 ± 0.2 at 4 h pi and 2.7 ± 0.2 at 24 h pi.These values are comparable with the data for the [ 177 Lu]Lu-DOT-AGA-PEG2-RM26 (2.8 ± 0.6 at 1 h pi and 3.1 ± 0.3 at 24 h pi) that in preclinical level demonstrated its utility to GRPR targeting therapy without development of nephrotoxicity (Mitran et al. 2019).The increase of tumor-to-kidney absorbed dose ratio might improve therapeutic potential of [ 177 Lu]Lu-AU-SAR-M1.Three strategies are proposed in literature for such improvement: i. structural modification of peptide-conjugate, ii.chemically-induced reduction of renal uptake, and iii.drug-induced reduction of radiation damage to the kidney (Geenen et al. 2021).Structural modification of small-size peptides may induce drastic changes in their biodistribution profile, as demonstrated in the present study.Moreover, interventions leading to overall charge changes of the peptide-radioligand may negatively affect GRPR-binding affinity.Alteration of biological properties may be caused as well by simple switch of radiometal used for labeling (Lymperis et al. 2018).It should be noted that such a radiometal switch, is the next phase in our studies, namely the exchange of the diagnostic SPECT radionuclide In-111 to its radiotherapeutic counterpart Lu-177.According to previous experience, the transition from In-111 to Lu-177 may lead to comparable or even superior biodistribution profile (Mitran et al. 2019;Mitran et al. 2016).Further pharmacokinetic improvements may occur during in situ NEP-inhibition, as previously reported for [ 177 Lu]Lu-DOTAGA-PEG2-RM26 (Mitran et al. 2019).In animals treated with the potent NEP-inhibitor phosphoramidon, the tumor-to-kidneys ratio increased to 3.8 ± 0.9 from 2.7 ± 0.4 in control group due to an increase in tumor uptake.We herein have shown that NEP-inhibition could induced using a fully characterized, commercially available registered drug (e.g.Entresto ® ) (Gu et al. 2010;McMurray et al. 2014)), facilitating clinical translation.Further studies to validate these prospects in animal models are currently on the way aiming for eventual proof-of-principle assessments in prostate cancer patients.Interestingly, several interventions for reduction of renal uptake, such as lysine/arginine or gelofusine infusion, as well as careful adjustment of the optimal injected peptide dose/ amount have been successfully used in the clinic (Geenen 2021).

Conclusions
Among the three new [ 99m Tc]Tc-DB15-base analogues designed to accommodate trivalent radiometals for theranostic use, [ 111 In]In-AU-SAR-M1 turned out to be the most promising, owing to its high GRPR-specific tumor uptake and retention in combination with a low and declining uptake in healthy tissues.The in situ stabilization of the radioligand in the blood-stream using Entresto ® (as a source of the potent and selective NEPinhibitor sacubitrilat), is expected to further upgrade the biodistribution profile of [ 111 In] In-AU-SAR-M1 and possibly its theranostic partner [ 177 Lu]Lu-AU-SAR-M1 as well.The renal uptake of [ 111 In]In-AU-SAR-M1 in absence of treatment with NEP inhibitor remained 2.5-fold lower than the tumor uptake at 24 h pi.This may still be the limiting factor for therapeutic applications in patients, potentially requiring NEP-inhibition and kidney protection regimens.Further studies are warranted to establish the radiotheranostic value of the [ 111 In]In-AU-SAR-M1/[ 177 Lu]Lu-AU-SAR-M1 pair, alone or during NEP-inhibition, in the management of prostate cancer or other GRPR-expressing malignancies.

Fig. 5
Fig. 5 Normalized in vitro cellular processing showing % of total cell associated activity (solid line) and fraction of activity internalized (dotted line) for a [ 111 In]In-AU-SAR-M1, b [ 111 In]In-AU-SAR-M2 and c [ 111 In] In-AU-SAR-M3 biodistribution results for [ 111 In]In-AU-SAR-M1, [ 111 In]In-AU-SAR-M2 and [ 111 In] Results are presented as average %IA/g ± sd, except for gastrointestinal tract and carcass, which are presented as average %IA ± sd.The biodistribution profiles of [ 111 In]In-AU-SAR-M1 and [ 111 In]In-AU-SAR-M2 were similar, while [ 111 In]In-AU-SAR-M3 showed more pronounced differences.Thus, the tumor uptake led to the following rank of analogues: [ 111 In]In-AU-SAR-M2 > [ 111 In] In-AU-SAR-M1 (p < 0.001) > > [ 111 In]In-AU-SAR-M3 (p < 0.0001 compared with the previous two).On the other hand, the highest kidney uptake was displayed by [ 111 In]In-AU-SAR-M3 (p < 0.05).Uptake in the GRPR-rich mouse pancreas was markedly higher for [ 111 In]In-AU-SAR-M2 (> fourfold higher than [ 111 In]In-AU-SAR-M1 and > 18-fold higher than [ 111 In]In-AU-SAR-M3; p < 0.0001 in both cases) and small intestines (p < 0.001 & p < 0.0001, respectively).With regards to liver uptake, [ 111 In]In-AU-SAR-M1 showed the lowest, but not significantly different, values compared with the other compounds.When T/O ratios are considered, [ 111 In]In-AU-SAR-M1 showed better (but not significantly higher) ratios for lungs, liver, muscle, bone and a lower ratio for pancreas in comparison with [ 111 In]In-AU-SAR-M3.In light of these results, [ 111 In]In-AU-SAR-M1

Fig. 8
Fig. 8 SPECT/CT images of a PC-3 tumor-bearing mouse, at a 4 h and b 24 h pi of [ 111 In]In-AU-SAR-M1, red arrows indicate tumor-sites Tc]Tc(O) 2 (N 4 )] +1 in [ 99m Tc]Tc-DB15 to [[ 111 In]In(DOTAGA)] −1 in [ 111 In]In-AU-SAR-M1 rendered [ 111 In]In-AU-SAR-M1 more vulnerable to the fast proteolytic action of the peptidase.Furthermore, introduction of L Arg next to DOTAGA in [ 111 In] In-AU-SAR-M2 improved its stability against NEP, whereas the presence of D Arg in

Table 1
Average labeling yield and in vitro stability tests in PBS and EDTA